The present invention relates to a novel human polypeptide defensin Def-X, homologous to HNP-4, its genomic DNA and cDNA.
The invention also relates to cloning and expression vectors, and cells transformed with said vectors. The subject of the invention is also the use of said polypeptides as antibiotic, cytotoxic, repair and endocrine regulatory agent and as pesticide as well as cosmetic or pharmaceutical compositions for the treatment of microbial infections, in particular bacterial, fungal and viral infections, or parasitic infections, cancers, of inflammation and of immune deficiency. Finally, the invention comprises diagnostic methods and kits for the determination of a microbial or parasitic infection and of an inflammation, or for screening for predisposition to immune deficiencies or cancer diseases.
Antimicrobial substances are key elements in the defence of multicellular organisms. Among these substances, there are both simple inorganic compounds (hydrogen peroxide, hypochlorous acid, nitric oxide) and complex proteins and peptides. They are present at the first lines of defence, at the surface of the mucous membranes of various organs, in particular in the epithelial cells of the intestine and of the lungs, depending on the species, as well as in the microbicidal organs of phagocytic cells of hematopoietic origin, where they were first identified. Their synthesis de novo or their release from storage sites—organelles of the lysosome or cytoplasmic granule type which are capable of storing them in an inactive or latent form—can be induced rapidly, which makes them particularly important in the early phases of resistance to infections (Martin et al., 1995).
The antimicrobial proteins of less than one hundred aminoacids in size are arbitrarily called antimicrobial peptides. Several families of antimicrobial peptides have been identified, which differ in the presence within them of disulfide bridges, in their aminoacid composition, their structural conformation and in their activity spectrum. The antimicrobial peptides comprising six conserved cysteines form the defensin family. This family is composed of antimicrobial peptides which are present in numerous species, which are abundant and which are about 3-4 kDa (Ganz and Lehrer, 1994). These peptides are formed of 30 to 40 aminoacids, of which six invariant cysteins which form three intramolecular disulfide linkages. They have complex conformation, are amphipathic, rich in beta antiparallel sheets but lack alpha helices (Lehrer and Ganz, 1992). The antimicrobial action of defensins is thought to result from their insertion into the membranes of the target cells, allowing the formation of voltage-dependent channels. White et al. (1995) describe the possible mechanisms of membrane insertion and of formation of multimeric pores by the defensins, which allow the permeabilization of the membranes of the target cells, for example microbial or tumor cells. The crystallographic structure of human neutrophil defensin HNP-3 (see below) has been determined, and a specific mechanism of dimerization of the human neutrophil defensins is in addition suggested. Increased knowledge of this family of peptides and comparison of their sequences and activity spectra will make it possible to better understand these mechanisms and their specificities, as well as the aminoacid residues more particularly involved in these phenomena.
The defensins are divided into three familities of peptides which are structurally different: the “conventional” defensins, the beta-defensins and the insect defensins. These families exhibit differences as regards the position of and the distance between the conserved cysteine residues, as well as those of other conserved aminoacids (proline, glycine) (Ganz and Lehrer, 1995).
Human defensins, of the conventional type, come essentially from two sources. They were first identified by peptide purification from neutrophil extracts. Four defensins have thus been isolated: “human neutrophil peptides” HNP-1, HNP-2, HNP-3, and HNP-4. The first three are different products of the same gene (Ganz et Lehrer, 1995). These three peptides represent 99% of the defensin content of the neutrophils, whereas HNP-4 is also present therein, but at concentrations which are 100 times lower. More recently, two human enteric defensins, HD-5 and HD-6, were characterized in the small intestine and more precisely in the Paneth cells (Bevins et al., 1996). While 16 enteric defensin genes have been identified in mice, only these two homologs have been identified in humans (Mallow et al., 1996).
Defensins have an antimicrobial action on a broad spectrum of microorganismes in vitro (Martin et al., 1995). This activity spectrum, which is particularly broad, comprises bacteria, Gram-positive and Gram-negative bacteria, several fungi, mycobacteria, parasites including spirochetes and several enveloped viruses including the HSV and HIV viruses. They are also cytotoxic for several categories of normal and malignant cells, including cells resistant to TNF-alpha and to the cytolytic NK factor (Kagan et al., 1994). The large quantity of targets of the defensins and their abundance in blood cells specialized in the immune defence, as well as the dramatic increase in their concentration during severe infections, suggest that these molecules could play an important role in the natural immunity to infections and to cancers. In particular, the increase in the transcription of the defensin genes and the release of cytoplasmic granules containing presynthesized defensins in response to stimuli, contributes to the local antimicrobial response, it being possible for the defensins to participate in the inflammatory reaction, in the repair processes and in endocrine regulation during infection. The hematopoietic defensins could contribute to the phenomenon of lysis of cancer cells, a phenomenon which is mediated by the neutrophils during the antibody-dependent immune response. The precise physiological role of the enteric defensins is not clearly established. They could stem the proliferation of the intraluminal flora or prevent the translocation of bacteria across the intestinal mucosa (Mallow et al., 1996). The abundance of the defensin mRNA in the Paneth cells reinforces the hypothesis that these epithelial cells could play a key role in the immune defence in the intestine. It has moreover been shown that their expression scheme coincides with the appearance of the Paneth cells during embryogenesis. Mallow et al. (1996) have suggested that low levels of expression of enteric defensins in the fetus could be the evidence of an immaturity of a local defence, which would predispose children born prematurely to infections due to intestinal microorganisms.
A defensin concentration corresponding to 10% of the normal level is observed in patients suffering from “specific granule deficiency”, a rare disease of the development of the granulocytes. The affected subjects suffer from frequent infections caused by common bacteria (Ganz and Lehrer, 1995).
Biochemically modified defensins are potential prophylactic and therapeutic agents against infections (Ganz and Lehrer, 1995). Research relating to these antimicrobial peptides or other molecules participating in the natural immunity have gained special importance since phenomena of resistance of microorgnisms to traditional antibiotics started to develop (Bevins et al., 1996).
The primary structure of defensins, in particular of human defensins, has been the subject of recent studies (White et al., 1995; Mallow et al., 1996). The conventional defensins comprise 29 to 35 aminoacids, but are derived from precursors—preproteins—comprising 90 to 100 aminoacids. The proteolytic maturation of the human neutrophil defensins to mature peptides is coupled with their despatch to the granulocytes; the function of the propeptide would include the inactivation of the precursor form of defensin and a support for the acquisition of the active conformation of the mature peptide (Martin et al., 1995). The peptide homologies are maximal at the level of the signal peptides, and minimal at the level of the mature peptides, which comprise nevertheless six cysteine residues which are fully conserved. While the conservation of these residues appears to be necessary for the acquisition of secondary structures which are involved in the activity of defensins, the differences in sequences which exist within the very large family of these antimicrobial peptides, in particular at their N-terminal end, but also in other nonconserved regions, appear to be important determinants of their activity spectrum, and of their antimicrobial or cytotoxic efficacy. The identification of novel members of this family of peptides, in particular of human defensins, is therefore necessary for understanding their mechanism of action and their specificity as well as for their use as anti-infectious and/or cytotoxic agents, or for designing variant peptides exhibiting specific spectra and/or of reduced or increased efficacy.
Sparkes et al. (1989) located the gene encoding HNP-1 on chromosome 8, in the 8p23 region. Bevins et al. (1995), and Mallow et al. (1996) lcoated the two genes encoding HD-5 and HD-6 on chromosome 8, more precisely in the 8p21-pter region, a region including the region previously identified as carrying the hematopoietic defensins. The genes encoding the human enteric defensins HD-5 and HD-6 contain two exons, whereas those encoding the hematopoietic defensins contain three of them, the last two exons encoding the prepropeptide, both in humans and in guinea pigs and rabbits (Mallow et al., 1996). Comparison of the genome sequences of the HD-5 and HD-6 genes has revealed a very strong similarity between the noncoding flanking sequences in 5′, suggesting that the latter contain the information necessary to the tissue specificity of the expression of these genes; these same regions carry, in addition, many binding sites for transcription factors, including two AP2 sites and six IL6 sites, suggesting pathways for regulating the expression of these genes during inflammatory processes. More generally, the very high degree of similarity between the sequences and the genomic organization of the defensins HNP-1, 2, 3, 4 and HD-5 and 6 led Bevins et al. (1995) to an evolutionary model attempting to relate the chromosomal organization of the family, and the homologous fractions of each pair of genes.
It is finally advantageous to note that the chromosomal region 8p23 is involved in numerous pathologies, in particular cancer pathologies: there may be mentioned, for example, hepatocellular carcinoma (Becker et al., 1996), non-small cell lung cancer (Sundareshan and Augustus, 1996), prostate cancer (Ichikawa et al., 1996), and colorectal carcinome (Yaremko et al., 1994). Although this has never been documented, it is possible that a deficiency in either of the human defensins has a role in the predisposition to such pathologies, or in their development.
The present invention relates to a novel human defensin, Def-X, which is homologous to defensin HNP-4.
The subject of the present invention is therefore an isolated polypeptide chosen from the following polypeptides:    a) polypeptide whose amino acid sequence is the sequence SEQ ID No. 3;    b) homologous, variant or modified polypeptide of the polypeptide whose amino acid sequence is the sequence SEQ ID No. 3;    c) polypeptide whose amino acid sequence is the amino acid sequence of a biologically active fragment of a polypeptide as defined in a) or b);    d) polypeptide comprising at least one fragment as defined in c).
In the present description, <<polypeptide>> will also be intended to designate a proteine or a peptide.
According to a preferred embodiment, the polypeptide according to the invention is characterized in that it consists of at least one of the following fragments:    a) signal peptide whose aminoacid sequence is the sequence SEQ ID No. 4, corresponding to the sequence between position 1 and position 19, ends included, of the aminoacid sequence SEQ ID No. 3;    b) proregion whose aminoacid sequence is the sequence SEQ ID No. 5, corresponding to the sequence between position 20 and position 63, ends included, of the aminoacid sequence SEQ ID No. 3;    c) mature peptide whose aminoacid sequence is the sequence SEQ ID No. 6, corresponding to the sequence between position 64 and position 94, ends included, of the aminoacid sequence SEQ ID No. 3; or    d) homologous, variant or modified fragment of a peptide according to a), b) or c).
Still preferably, the polypeptides according to the present invention correspond to the primary structure of the mature defensin defined above, that is to say the structure corresponding to the following aminoacid sequence SEQ ID No. 6                Ile Cys His Cys Arg Val Leu Tyr Cys Ile Phe Gly Glu His Leu Gly Gly Thr Cys        Phe Ile Leu Gly Glu Arg Tyr Pro Ile Cys Cys Tyrits homologs, variants or modified forms as well as their biologically active fragments and the polypeptides containing them.        
It is clearly understood that the polypeptides of the invention are in a nonnatural form, that is to say that they are not taken in their natural environment but that they may have been obtained by purification from natural sources or obtained by genetic recombination or by chemical synthesis as will be described below.
<<Homologous polypeptide>> is understood to mean a polypeptide whose aminoacid sequence exhibits at least 80%, and preferably 90%, of aminoacids in common.
<<Variant polypeptide>> is intended to designate a mutated polypeptide or a polypeptide corresponding to a polymorphism which may exist, in particular in human beings and which may exhibit a truncation, a substitution, a deletion and/or an addition of at least one aminoacid compared with the polypeptide according to the invention.
<<Modified polypeptide>> is understood to designate a polypeptide obtained by genetic recombination or by chemical synthesis as will be described below, exhibiting a modification relative to the normal sequence. These modifications may in particular apply to the pre, pro- or mature domains of the polypeptide according to the invention, the aminoacids responsible for a specificity of spectrum or of efficacy of activity, or responsible for the structural conformation, the charge or the hydrophobicity, and the multimerization and membrane insertion of the polypeptide according to the invention. It is thus possible to create polypeptides with equivalent, increased or reduced activity, and with equivalent, lower or broader specificity. The modifications may also apply to the sequences involved in the maturation, transport and addressing of the polypeptide.
<<Biologically active fragment>> of a polypeptide according to the invention is intended to designate a polypeptide fragment which has conserved at least one activity of the polypeptide from which it is derived, in particular:                capable of being recognized by an antibody specific for a polypeptide according to the invention; and/or        capable of acting as an antibiotic; and/or        capable of acting as an cytotoxic agent; and/or        capable of acting as an antitumor agent; and/or        capable of modulating tissue repair, endocrine regulation or the inflammatory process, in particular during an infection.        
According to the invention, the biologically active fragments of the polypeptides according to the invention will have a minimum of 10 amino acids, preferably 15 amino acids.
As has been indicated above, among the biologically active fragments, a preferred fragment is the mature peptide having the aminoacid sequence SEQ ID No. 6.
Among the homologs of the mature peptide, there should be mentioned the polypeptides in which up to 5 amino acids have been modified, truncated at the N- or C-terminal end, or deleted, or added, which represents about 80% of the sequence.
The biologically active fragments of this mature peptide preferably comprise from 10 to 15 aminoacids, the advantage of which may be being able to be easily obtained by chemical synthesis.
As indicated, the objective of the modifications of the mature polypeptide will be in particular:                to modulate the activity of the defensin,        to modify its specificity, both at the level of the microorganisms on which it is active and on its tissue localization,        to modify its bioavailability.        
The preceding compounds may be obtained using combinatorial chemistry, in which it is possible to systematically vary portions of the polypeptide before testing them on models, cell cultures or microorganisms, for example, in order to select the compounds which are most active or which have the desired properties.
Chemical synthesis also has the advantage of being able to use:                nonnatural aminoacids, or        nonpeptide bonds.        
Thus, to enhance the lifespan of the peptides, it may be advantageous to use nonnatural aminoacids, for example in D form, or aminoacid analogs, in particular sulfur-containing forms for example.
Finally, the structure of the mature defensin or of its homologs, variants or modified forms, as well as the corresponding fragments, may be integrated into chemical structures of the polypeptide type and the like. Thus, it may be advantageous to provide at the N- and C-terminal ends compounds not recognized by proteases.
The invention also comprises the nucleic acids encoding a polypeptide according to the invention.
According to a preferred embodiment, the nucleic acids according to the invention will be chosen from the following nucleic acids:    a) nucleic acid having the sequence SEQ ID No. 1 (genomic);    b) nucleic acid having the sequence SEQ ID No. 2 (cDNA);    c) equivalent, homologous, mutated or modified nucleic acid, compared with the nucleic acids according to a) or b);    d) fragments of the sequences a), b) or c) having at least ten bases;    e) nucleic acid capable of hybridizing with one of the sequences as defined in a), b), c) or d).
It is understood that the present invention does not relate to the genomic sequences in their natural chromosomal environment; they are sequences which have been isolated, that is to say that they have been recovered directly or indirectly, their environment having been at least partially modified.
They may thus be genomic DNA, cDNA or RNA comprising nonnatural nucleotides or not; they may also be isolated natural nucleic acids, or synthetic nucleic acids.
Equivalent nucleic acid will be understood to mean a nucleic acid encoding the polypeptides according to the invention, taking into account the degeneracy of the genetic code, and the corresponding cDNAs and RNAs.
Homologous nucleic acids will be understood to mean nucleic acid whose sequence exhibits at least 80%, preferably 90%, homology with the nucleic sequences according to the invention.
Mutated nucleic acid will be understood to mean any nucleic acid encoding a variant polypeptide according to the invention, and any nucleic acid comprising, compared with the sequences SEQ ID No. 1 and SEQ ID No. 2, at least one mutation in the promoter and/or regulatory sequences which may have an effect on the expression of the polypeptide, in particular on its level of expression and the tissue-specificity thereof. The sequences exhibiting a polymorphism which is present in human beings are therefore included in the invention. Among these polymorphisms, some may lead to immune deficiencies, in the response to infections, to predispositions to and/or to the development of cancers.
Modified nucleic acid will be understood to mean any nucleic acid encoding a modified polypeptide according to the invention, or any nucleic acid obtained by mutagenesis according to techniques well known to persons skilled in the art, and comprising modifications relative to the normal sequences, in particular mutations in the regulatory and/or promoter sequences, in particular leading to a modification in the level and/or the tissue-specificity of the expression of the polypeptide.
The present invention relates to all the primers and probes, which may be labeled according to methods well known to persons skilled in the art, which make it possible to identify, in particular by techniques based on hybridization or on amplification, for example by PCR, the nucleic sequences according to the invention, including discriminating between the normal sequences and the mutated sequences. Among the nucleic acid fragments of interest, there should be mentioned in particular the antisense oligonucleotides, that is to say whose structure ensures, by hybridization with the target sequence, inhibition of the expression of the corresponding product. The sense oligonucleotides should also be mentioned which, by interacting with proteins involved in the regulation of the expression of the corresponding product, will induce either an inhibition, or an activation of this expression.
They may be sequences which act both at the level of the exon or intron sequences described and on the flanking sequences, in particular the promoters and/or 5′ UTR regions.
The present invention also relates to cloning or expression vectors comprising a nucleotide sequence as described above.
These cloning or expression vectors may comprise elements ensuring expression of the sequence in a host cell, in particular promoter sequences and regulatory sequences which are effective in said cell.
It being possible for the vector in question to be autonomously replicating or to be intended to ensure the integration of the sequence within the chromosomes of the host cell.
In the case of autonomously-replicating systems, depending on the host cell, either prokaryotic or eukaryotic, systems of the plasmid type or viral systems will be preferably used, it being possible for the vector viruses to be in particular advenoviruses (Perricaudet et al., 1992), retroviruses, poxviruses or herpesviruses (Epstein et al., 1992). Persons skilled in the art know the technologies which can be used for each of these viruses.
Thus, it is known to use, as viral vector, defective viruses whose culture is carried out in complementation cells, this avoiding the possible risks of prolifeation of an infectious viral vector.
When the integration of the sequence into the chromosomes of the host cell will be desired, it will be necessary to provide, on either side of the nucleotide sequence to be integrated, one or more sequences derived from the host cell in order to ensure the recombination. These are also methods which are widely described in the prior art. It will be possible, for example, to use systems of the plasmid or viral type; such viruses will be, for example, retroviruses (Temin, 1986) or the AAVs, Adenovirus-Associated Virus (Carter, 1993).
The invention also relates to the prokaryotic or eukaryotic cells transformed with a vector as described above in order to ensure the expression of a natural, normal or variant, or modified defensin Def-X, or for example, of one of its fragments.
As indicated above, the present invention also relates to the polypeptides obtained by culturing the cells thus transformed and recovering the protein expressed, it being possible for said recovery to be carried out intracellularly or extracellularly in the culture medium when the vector has been designed to ensure the secretion of the protein by means, for example, of a “signal” sequence, the polypeptide being in the form of a pre-polypeptide or prepro-polypeptide. The constructs allowing the secretion of the polypeptides are known, both for prokaryotic systems and for eukaryotic systems. In the context of the present invention, some of the polypeptides Def-X may comprise their own secretory or membrane insertion system.
It is clearly understood that the recombinant polypeptides according to the invention may be obtained in glycosylated or nonglycosylated form and may have the natural tertiary structure or not.
Among the cells which can be used for the production of these polypeptides, there should of course be mentioned bacterial cells (Olins and Lee, 1993), but also yeast cells (Buckholz, 1993), as well as animal cells, in particular mammalian cell cultures (Edwards and Aruffo, 1993) but also insect cells in which methods using baculoviruses, for example (Luckow, 1993), may be used.
The cells thus obtained can make it possible to prepare natural, variant or modified polypeptides, Def-X, but also fragments of these polypeptides, in particular polypeptides which may correspond to the biologically active fragments.
The present invention relates, in addition, to the same polypeptides according to the invention but which are obtained by chemical synthesis and which may comprise nonnatural or modified aminoacids.
The polypeptides according to the present invention, in particular mature defensin, as well as the homologs, derivatives or modified mature polypeptides, may be obtained by chemical synthesis using any one of the numerous peptide syntheses known, for example the techniques using solid phases or techniques using partial solid phases, by condensation of fragments or by a conventional synthesis in solution.
When the compounds according to the present invention are synthesized by the solid phase method, the C-terminal aminoacid is attached to an inert solid support and comprises groups protecting its amino group at the alpha position (and if necessary, protections on its functional side groups).
At the end of this step, the group for protecting the amino terminal group is removed and the second aminoacid, which also comprises the necessary protection, is attached.
The N-terminal protecting groups are removed after each aminoacid has been attached; on the other hand, the protection is of course maintained on the side chains.
When the polypeptide chain is complete, the peptide is cleaved from its support and the protective side groups are removed.
The solid phase synthesis technique is described in particular in Stewart et al. (1984) and Bodanszky (1984).
The details of the synthesis will not be mentioned here; it should simply be recalled that the protective groups which are preferred for the alpha-amino groups are protective groups of the urethane type (BOC or FMOC). As regards the coupling reagents, they are very numerous; among them, there should of course be mentioned more particularly N,N′-diisopropylcarbodiimine (DIC) which is used in general in DMF or DCM.
When it will be desirable to use nonnatural aminoacids, it may be necessary to provide other types of reagent and in particular other types of protection system.
The present invention also relates to polyclonal or monoclonal antibodies obtained by immunological reaction in a human or animal body with an immunogenic agent consisting of a polypeptide according to the invention, in particular a polypeptide obtained by culturing one of the cells which have just been described, or by chemical synthesis as indicated above.
The invention therefore extends to the monoclonal and polyclonal antibodies or one of their fragments, chimeric antibodies, which are capable of specifically recognizing a polypeptide according to the invention.
The invention also comprises the antibodies according to the invention, characterized in that they labeled.
The labeled antibodies may be, for example, immunoconjugated with enzymes such as peroxidase or alkaline phosphatase, or labeled with the aid of fluorescent compounds, biotin or radiolabeled. The labeling techniques are well known to persons skilled in the art and will not be developed in the present description.
The invention also extends to the use of a polypeptide according to the invention as antimicrobial, in particular antibacterial, antifungal, antiviral and/or antiparasitic agent, as cytotoxic agent, in particular for anticancer use, and/or as agent for modulating inflammatory, tissue repair and endocrine, in particular corticostatic, regulating processes.
According to another aspect, the invention relates to a pharmaceutical composition comprising a polypeptide according to the invention, which may be combined with a pharmaceutically acceptable vehicle.
Such a composition may be administered by the systemic, local or topical route.
Its mode of administration, its dosage, its optimal galenic forms may be determined according to the criteria generally taken into account in establishing a treatment appropriate for a patient, in particular their age, their body weight, tolerance of treatment, its observed side effects, and the like.
The invention also comprises a pharmaceutical composition comprising a vector according to the invention which is capable of expressing in vivo a polypeptide according to the invention, which may be combined with a pharmaceutically acceptable vehicle.
It is also possible to envisage the expression of polypeptides or their fragments in vivo, in particular by means of gene therapy and using the vectors which were described above.
In the context of gene therapy, it is also possible to envisage the use of the sequences of the genes or of the cDNAs described above, <<naked>>, this technique was in particular developed by the company Vical, which has shown that it was, under these conditions, possible to express the polypeptide in some tissues without having recourse to the support of a viral vector in particular.
Still in the context of gene therapy, it is also possible to envisage the use of cells transformed ex vivo, which cells may then be reimplanted, either as such, or inside systems of the organoid type, as is also known in the state of the art (Danos et al., 1993). It is also possible to envisage the use of agents facilitating the targeting of a defined cell type, penetration into the cells or transport to the nucleus.
Said pharmaceutical compositions are, according to the invention, intended for the prevention and/or treatment of microbial infections, in particular microbial infections of bacterial, Gram-positive or Gram-negative bacteria, mycobacterial, fungal and viral origin, or parasitic, in particular spirochet, infections.
According to a preferred embodiment, the invention advantageously relates to the pharmaceutical compositions according to the invention, characterized in that the viral infections are infections linked to enveloped viruses, in particular the HSV and HIV viruses.
The subject of the invention is also pharmaceutical compositions according to the invention, intended for the prevention and/or treatment of cancers, in particular melanomas, liver, prostate or non-small cell lung cancer or colorectal carcinoma.
The invention comprises, in addition, pharmaceutical compositions according to the invention, intended to increase the immune defenses, to increase the immune defenses in the case of acquired immunodeficiency or to prevent the immunodeficiency, in particular for the treatment of psoriasis, or to modulate the inflammatory processes in cases in particular of chronic inflammatory diseases.
The polypeptides according to the present invention can be more particularly used in external topical form, for example on the skin and the mucous membranes. These external topical forms may be for pharmaceutical, dermatological or cosmetic use.
In particular, these compositions may be used as pharmaceutical antiseptic agent or as antiseptic in some cosmetics, either for cleansing the skin or superficial body growths and/or as preservative for the compositions.
The topical compositions according to the present invention may be used in particular in some skin, eye, vaginal or buccal conditions. They may also be used as additional cosmetic agent, in particular in some treatment shampoos.
The invention also relates to the detection of the absence or of an abnormal quantity of protein or of nucleic acid corresponding to defensin X as marker of an infection or of pathologies which will be described below.
The invention also relates to the detection of an abnormal form of the protein or the presence of an abnormal nucleic acid corresponding to a mutated defensin which may possibly be completely inactive. In this case, the presence of this abnormal form may be a marker of predisposition to certain conditions, in particular immunodeficiency and/or cancers.
Accordingly, the present invention relates to a method of diagnosing of an immunodeficiency and/or of a predisposition to certain types of cancer, characterized in that the presence of an abnormal defensin and/or of a sequence encoding an abnormal defensin is detected in a sample from a patient.
The diagnostic methods according to the present invention allow in particular the detection of an immunodeficiency and/or of a predisposition to one or more cancers, in particular those cited above, in particular in at-risk families. This type of diagnosis will in general be carried out by the detection of the mutated forms of the protein or of the nucleic acid sequences.
However, the invention also relates to methods for the diagnosis of inflammation, immunodeficiency, predisposition to conditions of the cancer type and/or infections due to microorganisms or linked to an immune deficiency or inflammatory phenomenon, characterized in that they comprise assaying a polypeptide or a nucleic acid according to the invention in a biological sample and comparing the result of said assay which is obtained with the quantity of polypeptide or nucleic acid normally present in an equivalent biological sample.
In this case, the peptide assay will allow, in general, detection of a microbial or parasitic infection and/or of an inflammation. The peptide assays may be carried out by any known method, ELISA or RIA for example. The detection of an abnormal form of defensin-X may be carried out, for example, with the aid of a monoclonal antibody which is specific for this form, in particular the antibodies which are the subject of the invention.
According to a preferred embodiment, the invention advantageously comprises the methods characterized in that they use an oligonucleotide probe and/or primer according to the invention.
The methods in which all or part of the sequence corresponding to the polypeptide Def-X is amplified beforehand by assaying nucleic acid according to the invention will be generally preferred, it being possible for these amplification methods to be carried out by the so-called PCR or PCR-like methods. PCR-like will be understood to designate all the methods using direct or indirect reproductions of the nucleic acid sequences, or in which the labeling systems have been amplified, these techniques are of course known; in general, they involve the amplification of DNA by a polymerase; when the original sample is an RNA, it is advisable to carry out a reverse transcription beforehand. There are currently very numerous methods which allow this amplification, for example the so-called NASBA “Nucleic Acid Sequence Based Amplification” (Compton, 1991), TAS “Transcription based Amplification System” (Guatelli et al., 1990), LCR “Ligase Chain Reaction” (Landegren et al., 1988), “Endo Run Amplification” (ERA), “Cycling Probe Reaction” (CPR), and SDA “Strand Displacement Amplification” (Walker et al., 1992), methods which are well known to persons skilled in the art.
The invention relates, in addition, to diagnostic kits or boxes for the determination of a microbial or parasitic infection, an inflammation, an immunodeficiency and/or a predisposition to cancer-type conditions, characterized in that they comprise an antibody according to the invention.
The diagnostic kits or boxes for the determination of a microbial or parasitic infection, an inflammation, an immunodeficiency and/or predisposition to cancer-type conditions, characterized in that they comprise a probe and/or a primer according to the invention are also included in the invention.
Finally, the subject of the invention is the use of a polypeptide according to the invention as pesticide, in particular for the cultivation of plants of industrial interest such as, for example, food plants such as corn, wheat, soybean, rice or rape, fodder plants, fruit trees, grape vine or ornamental plants.
Other characteristics and advantages of the present invention will emerge on reading the examples below, illustrated by the figures whose legends are described below.